The objective is to define the molecular event that mediate the regulation of aromatase activity in estrogen-producing cells. The factors that stimulate aromatase activity are: follicle-stimulating hormone, cyclic AMP analogues, androgens and estrogens in ovarian granulosa cells; cyclic AMP analogues and glucocorticoids in human adipose stromal cells. Phorbol esters potentiate the cyclic AMP induction of aromatase activity in human adipose stromal cells. In contrast, EGF inhibits cyclic AMP induction of activity in both cell types. By use of antibodies that we have raised against aromatase cytochrome P-450 (P-450AROM) we find that the cyclic AMP-mediated induction of aromatase activity of granulosa and adipose stromal cells is associated with an increase in synthesis of P-450AROM as well as an increase in the levels of its mRNA. We have used these antibodies to screen a Lambdagt11 human placental cDNA library and have isolated a 1.8 kb cDNA clone that encodes a long 3'-untranslated region and approximately 1.0 kb of coding sequence. This cDNA will be used to rescreen the Lambdagt 11 or a Lambdagt 10 cDNA library for isolation of a full-length cDNA clone (greater than 2.4 kb in length). The full-length cDNA will be characterized by restriction analysis and subcloned into bacteriophage M13 for sequence analysis. The cDNA probes will be used in hybridization studies to assess the effects of regulatory factors on the number of copies, rate of synthesis, processing and half-life of P-450AROM mRNA in estrogen-producing cells. The cDNAs will be used to screen a human genomic library for isolation of the P-450AROM gene, which will be characterized and partially sequenced. Chimeric genes that contain various lengths of 5' flanking region of the cytochrome P-450AROM gene fused to the structural region of the chloramphenicol acetyltransferase (CAT) gene will be transfected into human adipose stromal cells or HeLa cells in culture. The effects of regulatory factors on chimeric gene expression will be monitored by assay of CAT activity to determine which sequences are required for induction and repression of cytochrome P-450AROM gene transcription. Finally, the P-450AROM cDNA will be inserted into SV40 expression vectors and its synthesis expressed in COS1 and CV1 cells. The studies proposed will serve to elucidate the primary structure of cytochrome P-450AROM and its gene, to define the molecular mechanisms by which estrogen synthesis is regulated, and to begin to analyze the structure-function relationships concerning this protein.